Fig. 2. Gephyrin knock down reduces the clustering of various GABA_AR subunits to different extent.

A-F, Cultured hippocampal neurons were co-transfected with pEGFP-N1 and Geph CR (A, C, and E) or Geph CR3m (B, D, and F) shRNAs. Triple label immunofluorescence was done by using combinations of the mouse mAb to gephyrin and rabbit anti-α1, or rabbit anti-α2 GABA_AR subunit antibodies or the combination of rabbit anti-γ2 and the mouse mAb to β2/3. Note that the neurons transfected (EGFP fluorescence) with Geph CR (C and E) show a high reduction in the cluster density of gephyrin and α2, β2/3, and γ2-GABA_ARs when compared to neurons transfected with the control Geph CR3m (D and F). However, the reduction of α1 subunit containing GABA_AR clusters (A) is small when compared with Geph CR3m (B). G and H: Quantification of the effect of the gephyrin shRNAs on gephyrin and the GABA_AR cluster density. Values (mean±SEM) represent the percentages compared to the corresponding internal control (non-transfected sister cells in the same cultures). Significant differences with the corresponding internal control values are indicated by asterisks (**, p<0.01; ***, p<0.001 in Student's t-test). Comparisons between groups using one-way ANOVA Tukey test when compared at p<0.05 showed that the mutated shRNAs (Geph CR3m or Geph UTR3m) had no effect on the density of gephyrin and GABA_AR clusters. The cluster densities (number of clusters/100 μm²; mean ± SEM) in sister non-transfected cells in the Geph CR shRNA transfection experiments were 21.1±0.9 (632) for gephyrin, 20.9±0.9 (628) for γ2, 23.9±0.8 (717) for α1, 15.7±0.8 (472) for α2, and 17.7±0.8 for β2/3 (531) (The number of counted clusters is shown in parenthesis). Similar values for the non-transfected cells were obtained in the other transfection experiments. Arrowheads show colocalizing gephyrin and GABA_AR clusters. Scale bar: 5 μm.